1. Transactivator of Transcription Protein (Tat)
The human immunodeficiency virus (HIV) transactivator of transcription (Tat) protein is a pleiotropic factor that induces a broad range of biological effects in numerous cell types. The Tat protein is encoded by two exons near the center of the viral genome. The first exon encodes amino acids 1 to 72, and the second exon encodes amino acids 73 to 101, although naturally occurring Tat sequences may be up to 113 amino acids long and the first exon may be up to 86 amino acids long. Studies have indicated an 86 amino acid version of Tat is sufficient for its transactivation function, which is necessary for virus transcription and replication in vitro; this form is the form most commonly used for research investigations, which have largely focused on in vitro studies of Tat as a transactivator of viral transcription, as an immunoregulator and an inducer of apoptosis. These studies have essentially all been conducted in vitro at atmospheric oxygen levels, which studies leading to the current invention have shown to dramatically alter human peripheral blood leukocyte response to Tat.
The Tat protein contains several functional subdomains. The amino terminus (1 to 20), cysteine-rich domain (21 to 40), and core region (1 to 48) together constitute the minimal activation domain for transcription in vitro. The N-terminal portion of Tat binds cell surface antigen CD26 with high affinity and has been reported to be responsible for CD26-mediated immunosuppressive activity. The cysteine-rich domain has homology to chemokines and mediates binding to chemokine receptors. The basic domain (also known as “shlepper”), characterized by a high content of lysines and arginines, is required for binding to short RNA transcripts containing the viral transactivation-response element. This basic domain is essential for importing extracellular Tat and also binds to membrane proteins, including the vascular endothelial growth factor receptor and heparan sulfate proteoglycans. Free peptide corresponding to the basic domain of Tat translocates through the cellular membrane and accumulates in the nucleus. The basic domain also may mediate toxin-like properties of Tat, including neuronal toxicity, and it appears to signal through cyclic nucleoside phosphodiesterase 4 to alter cyclic AMP levels. The function of the C terminus is still uncertain, but may be necessary for pathogenesis in vivo, since primary isolates express Tat of greater than 101 amino acids. The C termini of most Tat variants also contains an RGD motif that mediates Tat binding to cell surface integrins. The length of Tat varies depending on virus strain or lade (meaning a taxonomic group comprising a common ancestor and all the descendants of that ancestor).
2. Roles of Tat
The Tat protein is a critical component in the mechanism of AIDS pathogenesis. It is found in both the nucleus of infected cells, where it serves a conventional role in virus transcription, and as a secreted protein that can bind to the cell surface through electrostatic interactions, chemokine receptors, or cell surface integrins. Rapid uptake and importation of Tat into the nucleus occurs in many different cell types; however, some biological effects of Tat may require only membrane binding because they occur below the concentrations needed for transactivation of nuclear gene expression.
Tat protein is a key regulatory protein required for production of viral RNA and viral replication. The Tat protein of HIV is a powerful transactivator of gene expression. By interacting with a structured RNA sequence at the 5′ end of the viral mRNA, it promotes the remodeling of chromatin and the recruitment of processive RNA polymerase complexes at the viral promoter. In addition to these transcriptional functions, a short amino acid motif (shlepper), highly enriched in basic amino acids (arginine rich domain), promotes the export of the protein from the expressing cells. Once in the extracellular environment, the same basic domain of Tat binds to cell surface heparan sulfate proteoglycans; apparently through this interaction, the protein is internalized by a variety of different cell types. Cellular internalization of Tat and Tat fusion proteins requires the integrity of cell membrane lipid rafts and mainly occurs through caveolar endocytosis. The Tat basic domain (shlepper), when attached to large protein cargos, also mediates efficient cellular internalization of these large protein cargos and thus can be utilized for transcellular protein transduction. This property already has been exploited successfully for the delivery of heterologous proteins, nanoparticles, liposomes, phage and viral vectors, and plasmid DNA. The biological significance of intercellular Tat trafficking in the context of viral infection still remains elusive.
Functional studies with Tat (summarized below) have largely been conducted in vitro, exclusively in standard incubators that are maintained in equilibrium with air (i.e., at atmospheric oxygen levels). Under these conditions, which are the current norm, the cells encounter oxygen levels well above the levels encountered in vivo (20% oxygen versus the 2% to 10% encountered in vivo). In addition, many of the studies conducted with Tat were conducted with long-established cell lines that have been grown exclusively at atmospheric oxygen levels and have lost many of the properties and sensitivities characteristic of peripheral blood mononuclear cells (PBMC) recently harvested from blood. In contrast, the studies that led to the current invention were conducted at physiological oxygen levels with freshly isolated PBMC, at which Tat behaves quite differently, e.g., it does not induce the extensive apoptosis that is induced at atmospheric oxygen levels. With this caveat, the summary below is presented.
Cell lines treated with Tat have shown increased expression of chemokine receptors, lower T-cell responses to antigenic stimulation, overproduction of interferon-α, and enhanced HIV replication due, it is thought, to increases in HIV transcriptional activation. Tat also has been shown to suppress mitogen-, alloantigen-, and antigen-induced lymphocyte proliferation in vitro by stimulating suppressive levels of interferon-α and/or by inducing extensive apoptosis. Tat may trigger apoptosis directly by induction of caspase pathways, or may increase expression of apoptosis inducing molecules (e.g., CD95 or TRAIL). Extracellular Tat also promotes T cell destruction (at atmospheric oxygen levels) by increasing expression of CD95L/Fas ligand on monocyte/macrophages and sensitizing cells to the effects of this molecule. CD95, also called Fas or APO-1, is a death receptor of the apoptotic mechanism and is expressed in activated T cells and NK cells. Other Tat studies have shown that there is an extensive loss of intracellular glutathione and production of ROS in Tat treated or transfected cells, perhaps due to Tat induction of oxidative stress (at atmospheric oxygen levels). Tat has been shown to facilitate Human Herpesvirus 8 (“HHV8”) infection of epithelial cells that can host the virus. Tat also has been known to participate in vivo in the HHV8 infection that leads to the induction of Kaposi's Sarcoma, a tumor caused by HHV8. Further, Tat has been known to induce oxidative stress (a change in the normal redox state).
Many studies have demonstrated that Tat efficiently induces apoptosis in a wide variety of cells, including cultured peripheral blood mononuclear cells (PBMCs). These studies have been conducted in cells cultured in standard CO2 (5%) incubators equilibrated with air (21% O2). This has led to suggestions that Tat could play a role in the development of HIV-related neuropathies in vivo. In vivo, Tat may be taken up by neighboring cells. In vitro, Tat isolated from viral and recombinant sources has been shown to enter uninfected cells through a temperature dependent endocytic pathway, which originates from cell membrane lipid rafts and follows caveolar endocytosis. The arginine motif (shlepper or transducer motif) at amino acid positions 49 to 57 (--49RKKRRQRRR57--: SEQ ID NO: 3), which transports Tat into cells, is sufficient to also transport other proteins and molecules into PBMC and cell lines. Thus, this motif commonly is incorporated into constructs or otherwise coupled to macromolecules to enable their ready entry into cells. Full-length Tat also can function as a transporter, although such a use of Tat tends to induce substantial apoptosis under typical culture conditions.
Studies that led to the present invention began by demonstrating that Tat does not induce apoptosis in PBMC cultured at physiological oxygen levels. Instead, culturing PBMC at these oxygen levels induces cell division in a proportion of the cells and results in priming the cells for subsequently in vitro infection with HIV. These findings were interpreted as revealing a novel role for Tat in HIV disease, i.e., indicating that Tat released in vivo by HIV infected cells may prime neighboring cells for infection and hence facilitate the spread of the HIV infection, particularly at the early stages of the disease.
3. Tat Immunogens
Despite the growing knowledge of HIV disease progression, there remains a need in the art for development of compositions and methods of treatment of HIV, that could slow the spread of the virus and possibly prevent the onset of the subsequent AIDS disease.
The potential for therapeutic or preventive immunization with Tat protein has been the subject of animal and clinical studies. Development of vaccines against Tat may allow for control of its toxic properties. Immunization studies in animals involving biologically active Tat or recombinant vaccinia vectors expressing Tat and Rev (anti-repression trans-activator protein) proteins have shown lower virus burden after challenge. In addition, the presence of anti-Tat serum antibodies or Tat-specific cytotoxic lymphocyte responses have been correlated with slow progression in HIV-infected individuals. However, the potential value of Tat as a vaccine antigen is controversial. Studies indicating complete or partial protection against viral challenge in macaques contrast with studies showing no protection. One study reported that therapeutic immunization with chemically inactivated Tat toxoid elicited strong immune responses in human beings that may be associated with clinical improvement. Other studies have shown that effective immunization with Tat toxoid failed to protect against mucosal transmission but did attenuate virus replication and disease. But although immunization was effective in protecting against disease, it did not provide sterilizing immunity against virus transmission. Response to immunization have been variable, but among animals with both cellular and humoral responses to Tat, studies have shown 88% were protected against disease progression.
Studies have indicated that Tat vaccines and, especially, formulations containing native Tat will not be effective as preventive, monovalent vaccines against HIV infection.
Additional studies utilizing modified inactive forms of the protein (Tat toxoid) via carboxymethylation of cysteine residues have been performed. Immunization studies with Tat or Tat toxoid have shown significant Tat-binding antibody titers and disease attenuation, although no animals were protected from infection. No statistically significant differences in disease among animals immunized with Tat or Tat toxoid in terms of viral RNA levels or CD4 cell counts were observed, although the Tat-immunized group tended to have lower viral RNA levels at set point compared with the Tat toxoid-immunized animals.
Studies utilizing monoclonal antibodies against the amino acid terminus or the domain RPPQ sequence have blocked Tat uptake into T cells and neutralized Tat in cell-based transactivation assays.
Several studies have focused on the role Tat plays in the immunopathogenesis of HIV, predicting that high-affinity neutralizing antibodies against Tat will improve the clinical prognosis of HIV infected patients. Epidemiological studies have observed an inverse relationship between the level of Tat-specific serum antibodies and the rate of disease progression. Studies indicate that the development of a highly effective Tat vaccine will depend on the identification of an appropriate Tat vaccine formulation that induces immune responses that completely block the immune-modulating activity of Tat.
HIV-1 Tat and Tat toxoid proteins are highly immunogenic in macaques and humans. Modified forms of Tat, including Tat toxoid, have been designed to avoid potential toxic effects of the protein. Studies have shown that these modifications restrict the pattern of antibody responses and elicit type-specific antibodies in macaques that do not recognize all Tat sequences equally. Thus, in order to study the response to Tat during infection or to develop broadly cross-reacting Tat vaccines, it is important to use sequences commonly present in the target population or to develop a mixture of antigens that overcomes the problem of sequence specificity.
Although animal models have been utilized to investigate Tat vaccine formulations, non-animal models are needed to accelerate the development of improved Tat vaccine formulations. The present invention addresses this need for non-animal models that allow design and pre-clinical assessment of HIV Tat vaccines.
Since Tat can prime cells for HIV infection under physiological conditions, monoclonal antibodies or other agents that can prevent Tat entry into cells or otherwise prevent Tat priming for HIV infection could be useful for slowing the progress of HIV disease.
To date, binding agents directed to the Tat molecule have proved unsuccessful in preventing entry of Tat into a cell. The present invention addresses this problem. It is based on the principle that, in order to prevent Tat priming, a binding agent, must block appropriate site(s) on the Tat molecule, and provides compositions and methods to identify such binding agents. The binding agents so identified may be used to identify targets on the Tat sequence that in turn may be used to produce vaccines that stimulate Tat inhibitory activity.